LMX1B Mutations Cause Hereditary FSGS without Extrarenal ...

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*Inserm U983, Hôpital Necker-Enfants Malades, Paris, France; †Service de ... J Am Soc Nephrol 24: 1216–1222, 2013. doi: 10.1681/ASN.2013020171 ... U983, 6e Etage, Tour Lavoisier, Hôpital Necker- ... them had CKD stage II–V, including 3 ... Figure 1. Patients with p.R246Q or p.R246P LMX1B mutations have FSGS ...
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LMX1B Mutations Cause Hereditary FSGS without Extrarenal Involvement Olivia Boyer,*†‡ Stéphanie Woerner,* Fan Yang,§ Edward J. Oakeley,| Bolan Linghu,§ Olivier Gribouval,* Marie-Josèphe Tête,* José S. Duca,§ Lloyd Klickstein,§ Amy J. Damask,§ Joseph D. Szustakowski,§ Françoise Heibel,¶ Marie Matignon,** Véronique Baudouin,†† François Chantrel,‡‡ Jacqueline Champigneulle,§§ Laurent Martin,|| Patrick Nitschké,‡¶¶ Marie-Claire Gubler,* Keith J. Johnson,§ Salah-Dine Chibout,| and Corinne Antignac*‡*** *Inserm U983, Hôpital Necker-Enfants Malades, Paris, France; †Service de Néphrologie Pédiatrique and ***Département de Génétique, Centre de Référence MARHEA, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France; ‡Université Paris Descartes, Sorbonne Paris Cité, Institut Imagine, Paris, France; §Novartis Institutes for Biomedical Research, Cambridge, Massachusetts; |Novartis Institutes for Biomedical Research, Basel, Switzerland; ¶ AURAL et Service de Néphrologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; **Service de Néphrologie, Hôpital Henri Mondor, Faculté de Médecine Paris XII, Assistance Publique-Hôpitaux de Paris, Paris, France; †† Service de Néphrologie Pédiatrique, Hôpital Robert Debré, MARHEA, Université Denis Diderot, Assistance PubliqueHôpitaux de Paris, Paris, France; ‡‡Service de Néphrologie et Médecine Interne, Centre Hospitalier de Mulhouse, Mulhouse, France; §§Service de Pathologie, CHU Nancy Brabois, Vandoeuvre, France; ||Service de Pathologie, et Inserm UMR1098, CHU Dijon, Dijon, France; and ¶¶Plateforme de Bioinformatique, Hôpital Necker, Enfants Malades, Paris, France

ABSTRACT LMX1B encodes a homeodomain-containing transcription factor that is essential during development. Mutations in LMX1B cause nail-patella syndrome, characterized by dysplasia of the patellae, nails, and elbows and FSGS with specific ultrastructural lesions of the glomerular basement membrane (GBM). By linkage analysis and exome sequencing, we unexpectedly identified an LMX1B mutation segregating with disease in a pedigree of five patients with autosomal dominant FSGS but without either extrarenal features or ultrastructural abnormalities of the GBM suggestive of nail-patella–like renal disease. Subsequently, we screened 73 additional unrelated families with FSGS and found mutations involving the same amino acid (R246) in 2 families. An LMX1B in silico homology model suggested that the mutated residue plays an important role in strengthening the interaction between the LMX1B homeodomain and DNA; both identified mutations would be expected to diminish such interactions. In summary, these results suggest that isolated FSGS could result from mutations in genes that are also involved in syndromic forms of FSGS. This highlights the need to include these genes in all diagnostic approaches to FSGS that involve next-generation sequencing. J Am Soc Nephrol 24: 1216–1222, 2013. doi: 10.1681/ASN.2013020171

disease (WT1, 3 LMX1B, 4 ACTN4, 5 TRPC6,6 INF2,7 ARHGAP248), some of which are associated with various types of extrarenal features. Indeed, heterozygous INF2 mutations that are responsible for most cases of AD FSGS7 may be also involved in Charcot-Marie-Tooth neuropathy–associated FSGS. 9 Mutations in the donor splice site of intron 9 of WT1 are responsible for Frasier syndrome 3 or isolated FSGS. 10 Finally, LMX1B mutations lead to nail-patella syndrome (NPS) (Online Mendelian Inheritance in Man [OMIM] #161200) associated with dysplasia of the patellae, nails, and elbows; iliac horns; glaucoma; and, in some cases, FSGS with specific Received February 20, 2013. Accepted March 27, 2013.

Primary FSGS is a clinicopathologic entity characterized by sclerotic changes affecting only a portion of some glomeruli, and glomerular symptoms consisting of isolated proteinuria or steroid-resistant nephrotic syndrome.1 During the past few years, the recognition of familial forms of FSGS has grown and the 1216

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identification of causative mutations in podocyte genes has been crucial in understanding the development and function of the glomerular filtration barrier.2 Although most genes have been identified in autosomal recessive forms, six genes have been involved in the rare autosomal dominant (AD) forms of the

Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Corinne Antignac, Inserm U983, 6e Etage, Tour Lavoisier, Hôpital NeckerEnfants Malades, 149 rue de Sèvres, 75015 Paris, France. Email: [email protected] Copyright © 2013 by the American Society of Nephrology

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lesions characterized by type III collagen fibrils in the glomerular basement membrane (GBM) on electron microscopy.4,11,12 However, mutations in these genes explain only a portion of the cases (approximately 20%).13 This list of genes will thus probably rapidly increase because of the high genetic heterogeneity observed and the recent availability of exome sequencing. Over the past decades, we have established a worldwide cohort of patients with hereditary FSGS, including 74 families with an apparent AD inheritance and no mutation in currently known FSGS genes. Among them, a large pedigree of FSGS and no extrarenal manifestation (family F, Figure 1) was of particular interest because it consisted of 10 affected patients (5 with available DNA) over 4 generations, with available DNA for 2 unaffected relatives (Figure 2A). Their clinical phenotypes are summarized in Table 1. The two index patients (F.III:3 and 6) presented with nephrotic-range albuminuria at 6 and 26 years of age and FSGS not otherwise specified on biopsy,1 with normal renal function at diagnosis. Patient F.III:3 progressed to ESRD by age 54 years. Electron microscopy was available for patient F.III:6 and did not show the specific anomalies of the GBM suggestive of nail-patella–like renal disease. Her son (patient F.IV:1) was tested positive for microalbuminuria (60 mg/d) at age 7 years during familial screening. The father of the index patients received a kidney transplant at age 60 years for ESRD of unknown origin. Six other relatives had various degrees of glomerular proteinuria detected between 22 and 70 years of age. Five of them had CKD stage II–V, including 3 who had reached ESRD at 36, 40, and 70 years of age. Two of them had biopsyproven FSGS. Electron microscopy was not performed except in patient F:III:6. None of the patients had any obvious extrarenal symptom. Direct sequencing of INF2 revealed no mutation in the 2 index cases. To identify the causal variant in family F, we first performed linkage analysis in the five affected individuals (patients F. II:3, F.III:1, F.III:3, F.III:6, and F.IV:1). J Am Soc Nephrol 24: 1216–1222, 2013

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Figure 1. Patients with p.R246Q or p.R246P LMX1B mutations have FSGS without extrarenal involvement. (A) Trichrome staining of kidney sections from patient V.II:1. Segmental sclerotic lesions are observed in the two glomeruli along with tubulointerstitial fibrosis. Original magnification, x80. (B) Electron microscopy of kidney sections from patient F.III:6. The glomerular basement membrane has a regular thickness and normal structure, and no specific ultrastructural change usually observed in NPS. Original magnification, x6500. (C) Photograph of the right hand of patient F.III:6. Nails display no features of dysplasia, such as triangular lunulae, longitudinal nail ridging, or splitting. (D–F) Radiographs of the elbows (D), pelvis (E), and knees (F) of patient V.II:1. No elbow dysplasia, radial head hypoplasia, iliac horn, patella dysplasia, or hypoplasia of the lateral femoral condyle can be noticed.

The maximum Lod score was 1.2. Seventeen regions of interest with a Lod score .1 were identified on chromosomes 1, 2, 3, 4, 6, 9, 11, 15, 17, and 19, spanning a total of 118 Mb (Figure 2B). This analysis excluded mutations in TRPC6 and ACTN4 in the family. Given the large number of positional candidates, we then performed

exome sequencing using next-generation sequencing in the five affected patients and in one of their unaffected relatives (subject F.II:7). The mean sequencing coverage was 86 reads. Nearly 96% of the SureSelect regions were sequenced at 153 depth or more, indicating nearcomplete coverage. LMX1B Mutations and FSGS

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A total of 315 variants common to the five individuals were identified within the regions of interest (Supplementary Table 1). After exclusion of all variants located in nonexonic regions (except splice sites), pseudogenes, untranslated regions, or known polymorphic variants, two variants were identified: one in the LMX1B gene (c.737G.A, p.R246Q) and the other in the HADHA gene (c.2026C.T, p.R676C). Upon inspection, the HADHA variant was eliminated as a candidate. HADHA mutations are responsible for long-chain 3-hydroxyacylcoenzyme A dehydrogenase deficiency (OMIM #609016) and mitochondrial trifunctional protein deficiency (OMIM #609105). These metabolic disorders involve the heart, muscles, and liver but not the kidneys, and both demonstrate recessive inheritance. Direct sequencing of LMX1B confirmed that the five affected patients were heterozygous for the p.R246Q, whereas the two unaffected relatives did not carry the mutation. We next sequenced LMX1B in a cohort of 73 additional unrelated families with podocytopathies of AD inheritance. The 91 patients with available DNA presented proteinuria at a median age of 24 years (range, 2–54 years), and FSGS was documented in at least 1 affected member in 55% of families. We identified the same p.R246Q mutation in a second family (family V). We also found a different mutation involving the same residue of the protein (p.R246P) in a third family (family B). Mutations segregated with the disease in both families (Figure 2A). The probands were diagnosed with nephrotic syndrome at 22 years and 5 years of age, respectively. Kidney biopsy specimens displayed FSGS not otherwise specified in family V and minimal-change disease in patients from family B. Both had a positive family history of minimal-change disease/FSGS in one of their parents. Until now, LMX1B mutations have been reported only in patients with NPS with or without renal involvement. Renal involvement has been reported in 2%–62% of cases of NPS14–17 and ranges from chronic benign proteinuria in most 1218

Figure 2. All identified mutations involve Arginine 246 of LMX1B. (A) Pedigrees of the three families that carry LMX1B mutations. The allele status is given below each tested individual. A heterozygous (het) mutation was detected in all affected persons. Family studies confirmed the segregation of the mutant allele as an AD trait. Normal individuals are represented by an open square or circle, depending on the sex, and affected individuals by a solid square or circle. Strikethrough represents deceased individual. (B) Linkage analyses in family F.

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Table 1. Spectrum of LMX1B mutations and associated phenotypes Family Patient Origin F

V

B

eGFR and Age at Last Follow-up

Predicted Age at Polyphen2 SIFT Nucleotide Effect on Exon a b Diagnosis Score Alterations Score Protein (yr)

I:2 II:1 II:3

Europe ND Europe ND Europe c.737G.A

ND ND p.R246Q

ND ND 4

ND ND 0.983

ND ND 0.02

40 ND 36

ESRD at 40 yr ND ESRD at 58 yr

II:6 II:8 II:9 III:1

Europe ND Europe ND Europe ND Europe c.737G.A

ND ND ND p.R246Q

ND ND ND 4

ND ND ND 0.983

ND ND ND 0.02

70 ND ND 22

III:3 III:6

Europe c.737G.A Europe c.737G.A

p.R246Q p.R246Q

4 4

0.983 0.983

0.02 0.02

6 26

ESRD at 70 yr CKD at 68 yr ESRD at 36 yr 61ml/min per 1.73 m2 at 59 yr ESRD at 54 years 120 ml/min per 1.73 m2 at 26 yr

IV:1

Europe c.737G.A

p.R246Q

4

0.983

0.02

7

I:2

Europe c.737G.A

p.R246Q

4

0.983

0.02

22

II:1

Europe c.737G.A

p.R246Q

4

0.983

0.02

17

I:1 II:1

Europe c.737G.C Europe c.737G.C

p.R246P p.R246P

4 4

0.993 0.993

0.04 0.04

25 5

115 ml/min per 1.73 m2 at 7 yr 88 ml/min per 1.73 m2 at 22 yr 45 ml/min per 1.73 m2 at 45 yr Normal eGFR at 55 yr 153 ml/min per 1.73 m2 at 17 yr

Kidney Histology

Extrarenal Feature

ND ND Terminal kidney ND ND ND FSGS

ND ND ND

FSGS FSGS; normal electron microscopy ND

None None

FSGS

None

FSGS

None

MCD MCD

None None

ND ND ND ND

None

All the mutations cited above have been found in the heterozygous state. Mutations numbering is based on the cDNA reference sequence (GenBank accession number NM_002316.3). ND, not determined; MCD, minimal-change disease. a A Polyphen2 score is predicted to be “probably damaging” if it is .0.85, “possibly damaging” if between 0.85 and 0.2, and “benign” if ,0.2. b A SIFT score ,0.05 is predicted to be deleterious, whereas a score $0.05 is tolerated.

patients to ESRD in 15%.14 Kidney histology displays nonspecific lesions on optical microscopy, mostly FSGS and consequences of renal failure.14–18 Characteristic ultrastructural changes are observed by electron microscopy in all patients, whether they have renal symptoms or not.14,18–20 They consist of focal or diffuse irregular thickening of the GBM containing patchy electron-lucent areas18 and irregular deposition of type III collagen fibrils, especially

in the lamina densa.16,21 Few cases of nail-patella–like nephropathy with ultrastructural changes of the GBM similar to lesions observed in NPS and without nail and osseous anomalies have been published,22–24 but the molecular bases of this phenotype have not been elucidated. Here, exome sequencing revealed a mutation in LMX1B segregating with disease in 3 of 74 families with isolated primary FSGS but no other known features of NPS.

Seventeen regions of interest spanning 118 Mb were identified on chromosomes 1, 2, 3, 4, 6, 9, 11, 15, 17, and 19 (with a maximum Lod score of 1.2). One 8.9-Mb region on chromosome 9, indicated here by an arrow, contains the LMX1B locus. (C) Three-dimensional representation of the LMX1B homeodomain. Left panel: Full view of the homology model of the two monomers of LMX1B (shown as yellow schematic) bound to a DNA fragment (orange schematic). The homeobox model only covers residues 218–275 of LMX1B because it is based on the x-ray template of the transcription factor NKX2.5 homeodomain dimer bound to the proximal promoter -242 site of the atrial natriuretic factor gene (ANF-242 DNA). The arginine 246 residue is depicted with blue spheres, in the predicted bound conformation to a DNA fragment, shown in stick representation (cyan and green carbon, blue nitrogen, red oxygen, and orange phosphate atoms). Right panel: Zoomed-in insert showing the predicted polar interactions (dotted yellow lines) between the R246 nitrogen atoms (shown in blue stick representation) and the ribose and phosphate oxygen atoms (red sticks) of DNA.

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Given this surprising finding, we contacted clinicians and referred the mutated probands for dermatologic, ophthalmologic, and examination of limbs and pelvis. Dysplasia of nails, patellae, or elbows; iliac horns; and glaucoma were definitively ruled out in all mutated probands. In addition, the electron microscopy specimen of patient F.III:6 was reprocessed and reanalyzed in the light of the LMX1B mutation to search for focal anomalies of the GBM suggestive of nail-patella–like renal disease. On the available sample, the GBM had a regular thickness and normal structure and no specific lesion usually observed in NPS. Although the patient probably had focal nail-patella–like renal lesions, this kidney biopsy specimen could not help elucidate the diagnosis. Although a stop-codon mutation in the arginine 246 has been reported in NPS,25,26 the two mutations involving R246 identified herein are novel mutations among the 164 LMX1B mutations reported to date.11,16,27,28 They cause LMX1B Mutations and FSGS

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nonconservative changes in a highly conserved amino acid. Polyphen2 and Sorting Intolerant From Tolerant (SIFT) scores for p.R246Q (0.983 and 0.04, respectively) and p.R246P (0.993 and 0.02, respectively) predict that the variants are probably damaging. None were referenced in the National Heart, Lung, and Blood Institute Exome Sequencing Project server displaying the data from 6503 control samples (http:// snp.gs.washington.edu/EVS/). LMX1B is a transcription factor belonging to the LIM-homeodomain family of proteins that plays an essential role in the normal development of dorsal limb structures, GBM, anterior segment of the eye, and some types of neurons. It contains two tandem zinc-binding LIM domains at the N-terminus involved in protein-protein interaction, a homeodomain in the middle responsible for DNA binding and necessary to transcriptional activation (in which R246 is located [Figure 2C], and a C-terminal glutamine-rich domain of unknown function.29 To make functional predictions, we mapped the mutated residue onto a LMX1B in silico homology model (Figure 2C). R246 in LMX1B is predicted to belong to a signature of interest containing a 5-residue random coil (SSKPC) followed by an 11-residue helix (RKVRETLAAET) common to several homeobox proteins. The homology model suggests that R246 plays an important role in strengthening the interaction between the homeobox domain of LMX1B and DNA, by holding interactions with the DNA. The guanidinium groups of R246 would hold a positive charge under physiologic conditions and would therefore be expected to hold strong electrostatic interactions with the negatively charged DNA backbone. This interaction would most likely be mediated via the DNA’s ribose and phosphate moieties.30 On the basis of this model, both mutations (p.R246Q and p.R246P) are expected to diminish such interactions and interfere with DNA binding. The mutation to a smaller and more hydrophobic residue-like proline may also disrupt the helical structure by adding a steric constraint.31 1220

Strikingly, Bongers et al. observed that patients with NPS who had mutations in the LMX1B homeodomain were more likely to exhibit nephropathy than those with mutations in other LMX1B domains.17 Our findings further emphasize and refine the relevance of the LMX1B homeodomain to renal function. Whereas the observations of Bongers et al. were made against a background of NPS, our findings were made in patients without extrarenal features, indicating that LMX1B arginine 246 has a kidney-specific function. On the basis of the homology model, LMX1B arginine 246 appears to play a role in stabilizing LMX1B/DNA interactions. Taken together, these observations suggest that LMX1B arginine 246 may be crucial for the interaction of LMX1B with its podocyte-specific downstream targets in the kidney, such as a3 and a4 type IV collagens, podocin and CD2AP.21,32–34 ChIP-seq experiments would be helpful to confirm this hypothesis. Moreover, this observation challenges, at least for some cases, the concept of haploinsufficiency currently proposed for LMX1B mutation dominant effect.35 In conclusion, we identified two novel mutations of the LMX1B gene in three unrelated families with AD FSGS and no extrarenal features. Our data demonstrate that isolated FSGS could be caused by mutations in genes also involved in syndromic forms of the disease and highlight the need to include these genes in all diagnosis approaches in FSGS. Moreover, these findings vividly illustrate the advantages that next-generation sequencing technologies offer in unraveling the genetic basis of human kidney disease.

CONCISE METHODS Genomic DNA specimens were isolated from peripheral blood using standard procedures. Written informed consent was obtained from participants or their parents, and the study was approved by the Comité de Protection des Personnes “Ile-De-France II.”

Linkage Analyses Genome linkage analysis was carried out on the 250k Affymetrix GeneChip 250k (NspI)

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single nucleotide polymorphism array platform (Affymetrix Inc.) in five affected individuals of family F. Data were evaluated by calculating multipoint Lod scores across the whole genome using MERLIN software, assuming AD inheritance with complete penetrance.36

Next-Generation Sequencing Analysis Exome enrichment was conducted at the Novartis facility using the Agilent SureSelect Human All Exon V4+UTRs capture kit. The multiplex libraries were then sequenced on an Illumina HiSeq2000 instrument with a 2376bp read length. Sequences were aligned to the reference human genome hg19 using the Burrows-Wheeler Aligner. 37 Downstream processing was carried out with the Genome Analysis Toolkit (GATK),38 SAMtools,39 and Picard, following documented best practices (http://www.broadinstitute.org/gatk/guide/ topic?name=best-practices). Variant calls were made with the GATK Unified Genotyper. All calls with read coverage of #2X or a Phred-scaled SNP quality score of #20 were removed from consideration. All variants were annotated using a software system developed by the Paris Descartes University Bioinformatics platform. Our analysis plan focused on identifying rare, protein-altering variants consistent with an AD inheritance pattern within the regions of interest identified by linkage analysis. We therefore assumed that the causal variant would satisfy the following criteria: The causal variant (1) belongs to the regions of interest identified by linkage analysis; (2) segregates perfectly with disease status; (3) is novel or is not referenced as a polymorphism (minor allele frequency . 1%) in databases such as dbSNP, the 1000 Genome Project, and an in-house exome sequencing variant database; and (4) alters a protein’s amino acid sequence and is likely to disrupt the function of that protein. Results were crossed with a parallel analysis performed by Novartis with a similar approach to determine potential pathogenic variants. The variant identified by exome sequencing was validated by Sanger sequencing, and segregation analysis in the family was verified. Sequence chromatograms were analyzed using the Sequencher software (Gene Codes, Ann Arbor, MI). Positions of mutations were numbered, with the A of the ATG-translation initiation codon in the reference cDNA

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sequence being 1. The functional consequence of each variant was predicted using SIFT (http://sift.jcvi.org/www/SIFT_enst_submit.html) and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/).

In silico Model The altered residue identified in the present study was mapped onto a three-dimensional homology model of LMX1B. The model was built with the Molecular Operating Environment suite of homology building tools developed by Chemical Computing Group Inc. The x-ray structure of the NKX2.5 homeodomain dimer bound to ANF-242 DNA (PDB accession code, 3rkq) was found to be the best structural match by the profile-profile comparison algorithms hhpred/hhseq.40,41 It contains a DNA fragment bound to a homeobox.

ACKNOWLEDGMENTS The authors wish to thank Anita Fernandez and Nicole Cheung for NGS data generation; NIBR IT for high-performance computing support; and Evan Beckman, Mark Fishman, Joanne Meyer, and Timothy Wright for strategic input. Financial support for this work was provided by grants from the Fondation pour la Recherche Medicale (Project DMP 2010-1120-386), the Agence Nationale de la Recherche (GenPod project: ANR-12-BSV1-0033.01), the European Community’s 7th Framework program grant, 2012-305608 (Eurenomics).

DISCLOSURES F.Y., E.J.O., B.L., J.S.D., L.K., A.J.D. J.D.S., K.J.J., and S.D.C. are employees of Novartis.

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See related editorial, “An Expanding Universe of FSGS Genes and Phenotypes: LMX1B Mutations Cause Familial Autosomal Dominant FSGS Lacking Extrarenal Manifestations,” on pages 1183–1185. This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi: 10.1681/ASN.2013020171/-/DCSupplemental.

J Am Soc Nephrol 24: 1216–1222, 2013